Enteroaggregative E. coli (EAEC) is an important diarrheal pathogen worldwide, and is the most common cause of bacterial gastroenteritis in the U.S. For 18 years we have explored the pathogenesis of this organism, including the description of a global activator of virulence genes, AggR, a multifunctional intestinal colonization factor, Aggregative Adherence Fimbriae (AAF), and a secreted protease called Pic, which cleaves mucin to provide supplemental growth substrates. In this request for continued funding, we will explore these and other aspects of EAEC pathogenesis. This application comprises four Aims. Aim 1. EAEC pathogenesis in the mouse. The IL10-/- mouse represents the first small whole animal model for EAEC. In this model, EAEC strain 042 elicits pan-colitis, which is not seen with other wild type E. coli strains tested. The streptomycin- treated conventional mouse, in contrast, serves as a colonization, but not disease, model, which we have used to characterize in vivo gene expression. In this aim we will use conventional mice and in vivo gene expression analyses to characterize the signals which control aggR expression in vivo, focusing specifically on biliary and pancreatic signals. We will also characterize the EAEC genes that contribute to colitis in the IL10-/- mouse, focusing on fliC, aafA and aafB mutants. Aim 2. Structure-function studies of the AAF adhesin. AAF are required for 1) binding of EAEC to the human intestine, 2) biofilm formation on abiotic surfaces, 3) adherence to T84 monolayers, 4) induction of IL-8 from polarized T84 monolayers, 5) disruption of tight junctions and barrier perturbation, and 6) adherence to and secretion of fibronectin. In this aim, we will leverage our recently completed solution structure of the AAF/II major pilin subunit to address the structure-function relationships underlying of these effects. Aim 3. Role of fibronectin binding and secretion in EAEC pathogenesis. We have shown that EAEC strains perturb the tight junctions of infected T84 monolayers, and also that EAEC induces the release of IL-8 from these epithelial cells. We have also found that EAEC binds to fibronectin, and that addition of Fn to T84 monolayers enhances adherence of 042 to the monolayer. Moreover, we have shown that infection of polarized T84 monolayers results in an increase in Fn release into the apical compartment. We will characterize the mechanisms of Fn release and the effects of Fn binding by EAEC. Aim 4. Role of the Pic protease in EAEC metabolism. Most EAEC strains and nearly all Shigella flexneri 2a strains secrete the Pic protease into the extracellular compartment. The presence of Pic enhances colonic colonization, and the mechanism of this effect in vitro appears to be via the release of growth substrates from mucin. In this aim we will characterize further this effect. Results of these studies will guide future interventions against this emerging pathogen.